Methyl benzoylformate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling: Triplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at
the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, triplicate samples from the test media of all test concentrations and the control (containing algae) were taken after 24 and 48h test duration and at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
All samples were diluted with acetonitrile. Therefore one part of the sample was diluted with nine parts acetonitrile.
Additional samples of the control blank and the dilution solvent were taken at test start and test end without any sample treatment.
The concentrations of the test item were analysed in two of the triplicate test media samples from all test concentrations, and in two of the triplicate control samples, from all sampling times (0, 24, 48 and 72 hours).

- Storage of Samples: All samples were stored in a freezer (≤ - 10 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 70.1 mg test item into 701 mL test water by intense stirring for 20 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other test concentrations. The test media were prepared just before introduction of the algae (= start of the test).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): The algae were supplied by the Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): The test was started (0 hours) by inoculation of a biomass of 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.
- Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.621 mmol/L as CO3 2-

Test temperature:

23 °C

pH:

7.5 to 8.0 at test start and 7.6 to 10.1 at test end

Nominal and measured concentrations:

100, 50, 25, 12.5 and 6.25 mg test item/L, corresponding to the initial mean measured concentrations of 73.0, 34.5, 15.0, 6.63 and 2.81 mg test item/L and a control.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium.
- Initial cells density: 5000 algal cells per mL test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (Reconstituted Water (OECD Medium))

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (OECD Medium)
- Alkalinity: 0.099 mmol/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The pH was measured in all test item concentrations and the control at the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media; Mean light intensity: 5113 lux (range: 4600 to 5630 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometrical measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not
replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of
the test media (with algae). Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline:
- Range finding study: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the
test solutions. In the pre-experiments, 100, 10, 1 and 0.1 mg test item/L were tested. No inhibition in growth was observed at 0.1, 1 and 10 mg test item/L. At 100 mg test item/L, a 24 % inhibition in yield and a 5 % inhibition in growth rate was observed in comparison to the control.
The quantification of the test item was performed using liquid chromatography with UV-vis detection (HPLC/UV-vis method).
At the start of the test recoveries of the nominal test concentration varied between 51 and 69% (all test concentrations considered). After 72 hours test duration, the recoveries of the nominal values varied between 34 and 3% (all test concentrations considered).
Analysis of two fortification levels showed a mean recovery rate of 78% (n = 4, RSD 7%).
In the developed quantification method a signal in the blank samples was detected. Therefore the results for the lower concentrations
could not be validated. Further development of the method resulted in another stabilisation of the samples and in a different chromatography. With these modifications it was possible to obtain reliable results.
The pre-experiments were not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the project number of the present study.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

94.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 92.4 - 96.5 mgIL (95 % conf. interval)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

68.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 67.0 - 70.3 mg/L (95 % conf. interval)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

56.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 52.4 - 59.8 mg/L (95 % conf. interval)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

39.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 36.4 - 42.0 mg/L (95 % conf. interval)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 39.1 - 42.4 mg/L (95 % conf. interval)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 25.1 - 27.6 mg/L (95 % conf. interval)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 6.35 - 7.63 mg/L (95 % conf. interval)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.24 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 2.89 - 3.60 mg/L (95 % conf. interval)

Details on results:

- Microscopic Examination: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a test concentration of nominal 100 mg test item/L and the algal cells in the control. Thus, the shape
of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

- Deviations to the Study Plan
Concerning: 2.5 Test Water, p. 10
According to Study Plan: Conductivity, water hardness and alkalinity will be given in the report.
Deviation to the Study Plan: The conductivity of the test water was not measured.
Reason for the Deviation: Human Error.
Presumed Effect on the Study: None.

Concerning: 2.10 Analysis of Test Item Concentration, HPLC-Conditions, p. 15
According to Study Plan: Mobile phase: acetonitrile/pure water
Deviation to the Study Plan: Pure water containing 0.1% H3PO4 was used as mobile phase B
Reason for the Deviation: Human Error.
Presumed Effect on the Study: None. Since all measurements were performed with this mobile phase.

Concerning: 2.10 Analysis of Test Item Concentration, Standard Solutions, p. 14
According to Study Plan: Aliquots of the stock solution will be diluted with appropriate solvent to get at least five standard solutions covering the range of samples’ analyte concentration ± at least 10 %, ideally ± 20 %.
Deviation to the Study Plan: The fortification level of 140 mg test item/L is slightly out of the calibration range.
Reason for the Deviation: Human Error.
Presumed Effect on the Study: Cause to the high accuracy of the fortification limit (mean recovery of 5 replicates 88%, RSD 2%) and the high linearity of the calibration curve (regression coefficient 1.0000) an extrapolation is justified.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The results of the most recent reference test are
EC50 (yield, 72 h) = 0.423 mg/L
EC50 (growth rate, 72 h) = 0.845 mg/L
EC50 (biomass, 72 h) = 0.434 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EC50 (growth rate) of the test material was determined to be 94.4 mg/l (nominal) under the conditions of the test.

Executive summary:

In this guideline (OECD 201) study conducted with GLP certification, the 72 hour EC50 (growth rate) of the test material was determined to be 94.4 mg/l (nominal). The test was conducted at five test concentrations (6.25, 12.5, 25, 50, & 100 mg/l nominal) in Pseudokirchneriella subcapitata under static conditions. Althought the test material was determined to be acutely harmful to the test organism, it does not meet the criteria for classification as hazardous to the aquatic environment, as it is readily biodegradable and has a low partition coefficient.

Description of key information

 Study carried out according to guideline and with GLP compliance.

Key value for chemical safety assessment

EC50 for freshwater algae:

68.6 mg/L

Additional information

Although the 72 hour algal EC50 (growth rate) for the test material is 94.4 mg/l (nominal), the test material is determined not to meet the criteria for Classification and Labelling as hazardous to the aquatic environment because the test material is readily biodegradable and has a partition coefficient (Log Pow) <4 (1.9 at 23 ºC).